PROJECT SUMMARY/ABSTRACT The midbrain superior colliculus (SC) receives extensive projections from sensory, motor and higher- association cortex, and projects downstream to brainstem motor nuclei. These cortico-tecto-brainstem projections are essential for attention-related motor eye movements. Despite this essential functional role and implications in attention-related disorders, little is known about the organization of cortico-tectal projections or their cell-type specific brainstem outputs. Neuroinformatics tools provide a computational capacity to analyze large-scale connectivity data in a reliable and feasible way to reveal novel connectivity organization networks. Previous studies reveal that cortical subnetworks topologically project to the striatum as functional domains. This suggests that cortico-tectal projections may also be organized into functional domains, and then descend to control downstream motor networks. Though classic tracers provide a wealth of input/output information of the SC, they cannot reveal synaptic specific connections. To reveal these connections and characterize their morphological properties, recently developed state-of-the-art rabies virus tracing methods and cutting-edge microscopy imaging provide new tools to elucidate specific pathways. The goal of this study is to construct a comprehensive wiring diagram of cortico-tecto-brainstem circuits that will be presented as an online resource to the scientific community. Specific Aim 1 will assemble a comprehensive connectivity projection map from the neocortex to the superior colliculus using classic anterograde/retrograde tracing techniques with neuroinformatics tools as part of the Mouse Connectome Project (www.MouseConnectome.org). This data will set an essential structural foundation to facilitate the study of specific attention-related circuits by revealing the organization of distinct functional subnetworks. Specific Aim 2 will define retrosplenial cortex (RSP) and anterior cingulate area (ACA) projections to distinct SC cell-types based on their anatomical locations, projection targets and neuronal morphologies. To define these cell types, this aim will use a monosynaptic rabies viral tracing method to map the synaptic connectivity between cortico-tectal inputs and brainstem- projecting SC neurons. The additional use of Lightsheet microscopy, SWITCH and 3D-reconstruction techniques utilized in pursuit of these aims render this project an ideal training venue. These studies will provide a more comprehensive foundation of cortical network organization within the SC that can also serve as a cross-species reference. Under the guidance of Dr. Hongwei Dong and colleagues in the Mouse Connectome Project, I will learn to perform high-quality histology, imaging and connectivity data analysis of SC neural networks. The mentorship and technical expertise I will acquire under this training grant will contribute greatly to my overall goal of becoming an independent neuroscientist studying detailed brain architecture.